Residual latent HIV provirus is predominantly integrated into introns of transcriptionally active genes of memory CD4+ T cells in virologically suppressed patients. Much of this provirus is subjected to transcriptional interference, where read-through transcription and splicing incorporate HIV RNA into mature cellular RNAs. We identified the inclusion of Tat first coding exon (Tat-exon2) within a cellular mRNA in latently infected cells, and low level expression of Tat mediated by an Internal Ribosome Entry Site (IRES) present in Tat-exon2. We have investigated the function of Tat IRES through investigation of the RNA structure and the cellular binding factors.
Suboptimal levels of Tat are expressed from the IRES-mediated translation of chimeric cell-HIV alternatively spliced read-through mRNA transcripts from adjacent cellular promoters. In an effort to identify new drugs that specifically reactivate latent HIV, but not other cellular genes, we developed reporter cell lines that recapitulated the RNA-processing pathways in HIV latency that lead to very low level Tat-expression from cellular-provirus read-through transcripts using the native IRES that underlies the first coding exon of tat. The 293.IRES CMV-CBG/LTR-CBR luciferase reporter cells included a second reporter that used a CMV-IE promoter to control for specificity. We screened 5,600 compounds in a known drug library and a library comprising of 114,000 drug-like compounds that identified 7 novel compound classes. Analogues of these 7 classes were examined in 11 point assay with CMV-CBG/LTR-CBR reporter cell lines and 106 compounds emerged giving a clear indication of early structure-activity relationships.
In conclusion, we found an important role for a conserved HIV RNA sequence-structure and several RNA binding proteins in Tat IRES translation from read-through cell-HIV mRNA transcripts. We successfully included this into a potential latency revering target that led to 7 novel classes of drug-like compounds warranting further development as HIV-specific latency purging drugs that specifically modulate HIV provirus in synergy with a low intrinsic expression of HIV RNA and Tat. We are iteratively developing our lead class for HIV-1 specificity and potency through medicinal chemistry and target analysis. We will also test for synergistic drug potency using cell line models, but also using primary HIV latent infected cells.